For cells to adapt to growth in a low-pH environment, such as a tumor, implies that the extrusion of metabolically generated intracellular protons must occur against an increased electrochemical gradient. Several plasma membrane proton translocation mechanisms have been described in various normal cells, and this project will use fluorescence technology for the measurement of intracellular pH (pH-i) in order to describe the low-pHe adaptation in terms of one or more of these. Transformed and non-transformed cell lines will be compared since the mechanism of cell adaptation to a low-pH environment is of particular interest with respect to cell growth in situ in tumors. Fluorescence measurements of pH-i using intracellular pH-sensitive probes (dyes) will be combined with selective inhibition of particular proton-motive mechanisms in order to assess the contributions of each to acid-base homeostasis. Besides total activity, molecular properties (such as the affinities of transporters for sodium or bicarbonate or inhibitors) will be compared. The hypothesis to be tested will be that cells which have adapted to growth in a low pH environment develop intrinsic metabolic or enzymatic differences which enhance their ability to their pH-i when placed in a medium with a lowered extracellular pHe. Functional differences to be measured include the rate at. which pH-i changes in response to inhibitors or stimulation by lowered pHi, since these may be more sensitive indicators of adaptation than simple measurements of static values of pHi in resting cells exposed to lowered pHe. In addi- tion, these will indicate which one(s) of the normal proton translocating mechanisms of the cell is enhanced or altered. Drugs to inhibit the adaptation may increase the therapeutic advantage of hyperthermia or other treatment modalities in vivo. Measurements will rely on whole-- spectrum fluorescence, rather than two-wavelength ratio methods. The fluorescence excitation spectrum of the probe BCECF will be used, and a second goal of the research win be to develop a fluorescence emission-spectrum method, based on the dye carboxy-SNARF-1, which will allow the use of adherent cell samples for single-cell measurements.